Work has continued on the characterization of a novel thyroxine transport protein found in human plasma, termed 27 K protein for its mobility in polyacrylamide gel electrophoresis under denaturing conditions. The protein was discovered as a contaminant of thyroxine-binding globulin (TBG) preparations. Further study revealed that 27 K protein binds one mole of thyroxine with Ka = 1.2 x 10 million as shown by equilibrium dialysis, tryptophan quenching and photoaffinity labeling. The affinity is much less than that of TBG and intermediate between those of prealbumin and albumin. The 27 K protein is immunochemically distinct from TBG as revealed by its lack of reaction with polyclonal anti-TBG serum absorbed with serum from TBG-deficient patients, and also by its reaction with monoclonal antibodies prepared against TBG and 27 K protein. Using human hepatoma cell cultures (Hep G2), 27 K protein was shown to be synthesized and secreted by liver cells and to be translated by mRNA separate from TBG mRNA. These studies also showed that, unlike TBG, K protein contains no carbohydrate. Further studies are required to clarify its physiological role in the transport of circulating thyroid hormones.